Method of demonstrating normal and pathological plasm-lipoprotein patterns in human body liquids

ABSTRACT

A method for the demonstration of normal and pathological plasmlipoprotein patterns in human body liquids by electrophoretical separation of the lipoproteins in a carrier medium, such as a gel, or on foils, which consists in that after the desired electrophoretical separation effect has been achieved the carrier is treated with a developing solution comprising polyanions, and an apparatus for carrying out the method.

States atent' 1 Seidel et a1.

[ 1 Mar. 25, 1975 [58] Field of Search 204/180 0, 299

[56] References Cited UNITED STATES PATENTS 3,766,047 10/1973 Elevitch 204/299 3,773,647 11/1973 Mandle et a1 204/299 3,783,118 l/1974 Hjerten v. 204/180 G Primary Examiner-John H. Mack Assistant Examiner-A. C. Prescott Attorney, Agent, or Firm-Steinberg & Blake 10 Claims, 6 Drawing Figures METHOD OF DEMONSTRATING NORMAL AND PATHOLOGICAL PLASM-LIPOPROTEIN PATTERNS IN HUMAN BODY LIQUIDS [75] Inventors: Dietrich Seidel; Heinrich Wieland,

both of Heidelberg, Germany; Johann Eibl, Vienna, Austria; Gerald' Eder, Vienna, Austria; Ewald Molinari, Vienna, Austria [73] Assignee: l mmuno Aktiengesellschaft Fur Chemisch medizinische, Vienna, Austria [22] Filed: Dec. 19, 1973 [21] Appl. No.: 426,202

[30] Foreign Application Priority Data Feb. 12, 1973 Austria 1218/73 [52] US. Cl. 204/180 G, 204/180 S, 204/299 [51] Int. Cl. B0lk 5/00 l i :8 f X 5 =1 PATENIEDmzsms 3 73 4:33

sum 1 p g METHOD OF DEMONSTRATING NORMAL AND PATHOLOGICAL PLASM-LIPOPROTEIN PATTERNS IN HUMAN BODY LIQUIDS The invention relates to a method of demonstrating normal and pathological plasm-liporprotein patterns in human body liquids by electrophoretical separation of the lipoproteins in a carrier medium, such as a gel, or on foils, and an apparatus for carrying out the method.

The analysis of the serum-plasm-lipoproteins is a prerequisite for an exact diagnosis of different forms of hyperlipoproteinernia and demands have been raised in the past for finding a reliable, easily practicable technique of identifying the normal and abnormal lipoprotein fractions. Demonstration methods are known that are based on the electrophoresis of human body liquids. Therein the lipoproteins of human. serum or the like are subjected to the influence of an electric field in a solid migration medium, mainly a gel, the migration of the lipoproteins causing a separation effect. The lipoprotein bands are made visible in the known method by coloring with lipid coloring agents or by immunological precipitations, e.g. with antiserum. In many cases, in combination with it, an ultracentrifugation is necessary; the known methods have the disadvantage that they require such a high expenditure of work and such costly apparatus that a complete lipoprotein analysis can be carried out in special laboratories only.

It is the object of this invention to provide a method which overcomes these disadvantages, which is practicable in any laboratory and guarantees reliable results.

The method of the invention, which starts from the electrophoretical separation of the lipoproteins in a carrier medium, such as a gel, or on foils, is characterised in that after the required electrophoretical separa tion effect has occurred, the carrier medium is treated with a developing liquid, which comprises one or several of the following substances: polyanions, such as heparin or dextran sulfate, sodium-dodecylsulfate (SOS), sodium-phosphotungstic acid, sodium oleate, sodium salts ofbile acids, suitably in the presence of bivalent cations, such as magnesium and calcium, wherein complex salts of low solubility are formed with the lipoproteins.

The chemical composition of the developing solution or the kind of polyanions to be used, respectively, depends upon the lipoproteins that are to be demonstrated.

For demonstrating the abnormal LP-X-lipoprotein suitably a developer, containing heparin, magnesium ions and sodium chloride, is used.

For demonstrating the abnormal lipoprotein type III, it is also suitable to use a developer containing heparin, magnesium ions and sodium chloride.

According to a modified embodiment of the invention, a carrier, e.g. a gel plate, is used for demonstrating the LP-X-lipoprotein, the carrier already containing the developing solution or parts of it.

If the lipoprotein type III, on the one hand, and the lipoproteins VLDL, LDL and HDL, on the other hand, are to be demonstrated selectively, the carrier is treated in turns with different developing solutions, the one containing heparin and magnesium ions and the other dextran sulfate and calcium ions.

Furthermore, the invention comprises an apparatus for carrying out the method of the invention, which, in a manner known, possesses a carrier in form of a plate bearing a layer of the migration medium, e.g. of the gel, said carrier being immersed into an electrolyte or in a buffer solution.

The apparatus of the invention comprises a row of slits or holes and possibly a device for bringing into touch the plate-formed carrier with the developing solution. 1

For demonstrating the LP-X-lipoprotein the row of holes or slits is suitably arranged on the side of the anode and for demonstrating the lipoprotein pattern VLDL, LDL, HDL and type III, the row is arranged on the side of the cathode.

The row of holes may however also be arranged in the center of the gel carrier having the form of a plate, in which case starting from this row, the lipoprotein LP-X migrates in the direction towards the cathode and the other lipoproteins in the direction towards the anode.

According to a preferred embodiment, the carrier, having the form of a plate, is U-shaped and insertable into an electrolyte or into a buffer solution receiving container, respectively, which is divided in longitudinal direction by a separating dam into an anode region and a cathode region.

The drawing shows the apparatus of the invention by way of an example.

FIG. 1 illustrates a vertical section of the apparatus and FIG. 2 is a top view of the gel carrier.

FIG. 3 (FIGS. 30 3d) shows representations of plasm lipoprotein patterns received arrording to the invention.

The apparatus comprises a rectangular vessel 1 which may be covered by a lid 2. Platinum sheets 3 and 4 or platinum wires, respectively, serve as cathode K and anode A which continue in the interior of the vessel along the side walls and along part of the bottom. Fillets 5 extend in upward direction from the bottom of the vessel; they represent a separating dam and divide the vessel 1 in longitudinal direction into the cathode and the anode region. The gel plate 7 rests on the fillets 5, thus bridging the separating dam and immerses into the buffer solution with its limbs 8 extending downwardly. The gel plate is cast into an about U-shaped stencil 9. The stencil is suitably made of plastics. In the gel plate on the side of the cathode a row of slits 10 is provided, which serves for receiving the human body liquid to be examined, e.g. the human serum. By applying an electric field, the lipoproteins migrate in the direction of the anode and are thereby separated into the individual fractions depending upon their migration speed. As soon as the required separation effect has been achieved, the stencil is removed from the apparatus and is immersed into a container (not shown) which holds the developing solution. It is however also possible to drop or spray the developing solution onto the stencil, thus making the individual lipoprotein bands visible after an appropriate period of influence.

EXAMPLE 1 For demonstrating the lipoprotein bands in the human serum, the electrophoresis was carried out in an agarose migration medium as described by R. I. Noble, I. Lipid Res. 9.693 (1968). A veronal buffer with a pH of 8.6 and with an ionic strength of 0.05 was used. After the electrophoresis had continued for an hour, the stencil was removed from the apparatus and put into a developing bath which consisted of 0.55 dextran-sulfate 2,000 and of a 2.2 aqueous calciumchloride solution. The carrier was allowed to stay in the bath for 15 to 20 minutes at room temperature. During that period a complete precipitation of the following lipoprotein bands occurred:

chylomicrons B-lipoproteins pre-[i-lipoproteins OZ-llPOPI'OIGlHS, which in FIG. 3a are denominated in turn with 1,2,3 and 4.

EXAMPLE 2 In accordance with example 1, type III human serum on an agar stencil was subjected to the electrophoresis in a veronal buffer having a pH of 8.6 and an ionic strength of 0.05 for the purpose of determining the hyperlipoproteinemia. After the electrophoresis had continued for an hour, the stencil was removed and put into a developing bath consisting of 0.25 heparin and 0.95 70 magnesium chloride in a 0.95 aqueous sodium-chloride solution and was allowed to stay in the bath for 20 minutes. The precipitation for the field VLDL, characteristic of type III was clearly to be recognised and is denominated with 5 in FIG. 3b. The field extends from the B-position 2 as far as the pre-B- position 3.

EXAMPLE 3 For demonstrating LPX-lipoprotein, four samples of human serum were inserted into the four holes of an object holder carrying an agar plate and by means of filter paper the object holder was connected with a veronal buffer solution, having a pH of 8.6 and an ionic strength of 0.05. The samples thus prepared were subjected to the electrophoresis. After the electrophoresis had continued for half an hour, the object holder was removed and put into a developing bath consisting of 0.25 heparin and 0.95 magnesium chloride in 0.9 aqueous sodium-chloride solution, and was allowed to stay in the bath for minutes. Two samples which are denoted with N in FIG. 3c were negative, in the other two positive samples, denoted with P, the LPX- band was easily recognisable and was denoted with 6.

EXAMPLE Human full plasm was subjected to a polyacrylamide-electrophoresis, according to the method by B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964), wherein a tris-glycin buffer [tris-(hydroxymethyl)- aminomethane-buffer] with a pH of 8.8 and an acrylamide-monomer concentration of 3.5/7 was used. When the required separation effect had been achieved after a two-hour electrophoresis, the glass tube was removed from the apparatus, the polyacryl-amide gel was removed and treated with a developing solution of the following composition: 0.55 dextran sulfate 2,000 and 2.2 aqueous calcium-chloride solution.

The following lipoprotein bands were thus made visible and were identified:

pre-B-lipoproteins B-lipoproteins a-lipoproteins, which are denoted with 3,2 and 4 in FIG. 3d.

What we claim is:

1. A method of demonstrating normal and pathological plasm-lipoprotein patterns in human body liquids which comprises electrophoretically separating lipoproteins in a carrier medium or on foils,

upon occurrence of a desired electrophoretical separation effect, treating the carrier with a developing solution, comprising at least one of the following substances: polyanions, selected from the group consisting of heparin and dextran sulfate, sodiumdodcyl -sulfate (SOS), sodium phosphotung stic acid, sodium oleate, sodium salts of bile acids, lowsoluble complex salts being formed with the lipo- 1 proteins.

2. A method according to claim 1, wherein the carrier medium is a gel.

3. A method according to claim 1, wherein bivalent cations, selected from the group consisting of magnesium and calcium, are additionally present in the developing solution.

4. A method according to claim 1, wherein for demonstrating LP-X-lipoprotein, a developing solution is used which comprises heparin and magnesium ions.

5. A method according to claim 4, wherein the developing solution additionally contains sodium chloride.

6. A method according to claim 1, wherein for demonstrating lipoprotein type III a developing solution is used which comprises heparin and magnesium ions.

7. A method according to claim 6, wherein the developing solution additionally contains sodium chloride.

8. A method according toclaim 1, wherein for selectively demonstrating lipoprotein type III and lipoproteins VLDL, LDL and HDL the carrier is successively treated with a first developing solution and a second developing solution, the first developing solution comprising heparin and magnesium ions and the second developing solution comprising dextran sulfate and calcium ions.

9. A method according to claim 1, wherein for demonstrating lipoprotein LP-X a carrier is used which already contains at least parts of the developing solution.

10. A method according to claim 9, wherein the carrier is a gel plate. 

1. A METHOD OF DEMONSTRATING NORMAL AND PATHOLOGICAL PLASMA-LIPOPROTEIN PATTERNS IN HUMAN BODY LIQUIDS WHICH COMPRISES ELECTROPHORETICALLY SEPARATING LIPOPROTEINS IN A CARRIER MEDIUM OR ON FOILS, UPON OCCURRENCE OF A DESIRED ELECTROPHORETICAL SEPARATION EFFECT, TREATING THE CARRIER WITH A DEVELOPING SOLUTION, COMPRISING AT LEAST ONE OF THE FOLLOWING SUBSTANCES: POLYANIONS, SELECTED FROM THE GROUP CONSISTING OF HEPARIN AND DEXTRAN SULFATE, SODIUM-DODECYL-SULFATE (SOS), SODIUMPHOSPHOTUNGSTIC ACID, SODIUM OLEATE, SODIUM SALTS OF BILE
 2. A method according to claim 1, wherein the carrier medium is a gel.
 3. A method according to claim 1, wherein bivalent cations, selected from the group consisting of magnesium and calcium, are additionally present in the developing solution.
 4. A method according to claim 1, wherein for demonstrating LP-X-lipoprotein, a developing solution is used which comprises heparin and magnesium ions.
 5. A method according to claim 4, wherein the developing solution additionally contains sodium chloride.
 6. A method according to claim 1, wherein for demonstrating lipoprotein type III a developing solution is used whiCh comprises heparin and magnesium ions.
 7. A method according to claim 6, wherein the developing solution additionally contains sodium chloride.
 8. A method according to claim 1, wherein for selectively demonstrating lipoprotein type III and lipoproteins VLDL, LDL and HDL the carrier is successively treated with a first developing solution and a second developing solution, the first developing solution comprising heparin and magnesium ions and the second developing solution comprising dextran sulfate and calcium ions.
 9. A method according to claim 1, wherein for demonstrating lipoprotein LP-X a carrier is used which already contains at least parts of the developing solution.
 10. A method according to claim 9, wherein the carrier is a gel plate. 